Structures of a P4-ATPase lipid flippase in lipid bilayers

AbstractType 4 P-type ATPases (P4-ATPases) are a group of key enzymes maintaining lipid asymmetry of eukaryotic membranes. Phospholipids are actively and selectively flipped by P4-ATPases from the exoplasmic leaflet to the cytoplasmic leaflet. How lipid flipping is coupled with ATP-hydrolysis by P4-ATPases is poorly understood. Here, we report the electron cryo-microscopy structures of a P4-ATPase, Dnf1-Cdc50 from Chaetomium thermophilum, which had been reconstituted into lipid nanodiscs and captured in two transport intermediate states. The structures reveal that transmembrane segment 1 of Dnf1 becomes highly flexible during lipid transport. The local lipid bilayers are distorted to facilitate the entry of the phospholipid substrates from the exoplasmic leaflet to a cross-membrane groove. During transport, the lipid substrates are relayed through four binding sites in the groove which constantly shields the lipid polar heads away from the hydrophobic environment of the membranes.

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Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

10.1101/2021.11.03.467174 ◽

2021 ◽

Author(s):

Thibaud Dieudonne ◽

Sara Abad Herrera ◽

Michelle Juknaviciute Laursen ◽

Maylis Lejeune ◽

Charlott Stock ◽

...

Keyword(s):

Atp Hydrolysis ◽

Eukaryotic Cell ◽

Binary Complex ◽

C Terminus ◽

Catalytic Sites ◽

Marked Preference ◽

Exogenous Compounds ◽

P Type ◽

Lipid Flippase ◽

Full Activation

P-type ATPases from the P4 subfamily (P4-ATPases) are primary active transporters that maintain lipid asymmetry in eukaryotic cell membranes by flipping lipids from the exoplasmic to the cytosolic leaflet. Mutations in several human P4-ATPase genes are associated with severe diseases. For instance, mutations in the ATP8B1 gene result in progressive familial intrahepatic cholestasis, a rare inherited disorder that usually progresses toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 angstrom resolution. The lipid flippase complex is autoinhibited by the N- and C-termini of ATP8B1, which in concert form extensive interactions with the catalytic sites and flexible domain interfaces of ATP8B1. Consistently, ATP hydrolysis by the ATP8B1-CDC50A complex requires truncation of its C-terminus as well as the presence of phosphoinositides, with a marked preference for phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminus further suggests molecular communication between N- and C-termini in the autoinhibition process and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A conserved (G/A)(Y/F)AFS motif in the C-termini of several P4-ATPase subfamilies suggests that this mechanism is employed widely across P4-ATPase lipid flippases, including both plasma membrane and endomembrane P4-ATPases.

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Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum

Biochemical Journal ◽

10.1042/bj3560685 ◽

2001 ◽

Vol 356 (3) ◽

pp. 685-704 ◽

Cited By ~ 78

Author(s):

Kathleen J. SWEADNER ◽

Claudia DONNET

Keyword(s):

Crystal Structure ◽

Sarcoplasmic Reticulum ◽

Binding Sites ◽

Atp Hydrolysis ◽

Structural Features ◽

Cleavage Sites ◽

Transmembrane Helices ◽

Transmembrane Topology ◽

Key Features ◽

P Type

The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca2+-ATPase) has recently been determined at 2.6 Å (note 1 Å = 0.1nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647–655]. Other P-type ATPases are thought to share key features of the ATP hydrolysis site and a central core of transmembrane helices. Outside of these most-conserved segments, structural similarities are less certain, and predicted transmembrane topology differs between subclasses. In the present review the homologous regions of several representative P-type ATPases are aligned with the SERCA sequence and mapped on to the SERCA structure for comparison. Homology between SERCA and the Na,K-ATPase is more extensive than with any other ATPase, even PMCA, the Ca2+-ATPase of plasma membrane. Structural features of the Na,K-ATPase are projected on to the Ca2+-ATPase crystal structure to assess the likelihood that they share the same fold. Homology extends through all ten transmembrane spans, and most insertions and deletions are predicted to be at the surface. The locations of specific residues are examined, such as proteolytic cleavage sites, intramolecular cross-linking sites, and the binding sites of certain other proteins. On the whole, the similarity supports a shared fold, with some particular exceptions.

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Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

Author(s):

Mateusz Dyla ◽

Sara Basse Hansen ◽

Poul Nissen ◽

Magnus Kjaergaard

Keyword(s):

Structural Dynamics ◽

Single Molecule ◽

New Technologies ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved ◽

Dynamics Simulations ◽

Types Of Information ◽

P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys693 and Asp714 in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/j.bbabio.2006.06.008 ◽

2006 ◽

Vol 1757 (11) ◽

pp. 1485-1495 ◽

Cited By ~ 22

Author(s):

Juha Okkeri ◽

Tuomas Haltia

Keyword(s):

Escherichia Coli ◽

Atpase Activity ◽

Metal Binding ◽

Binding Sites ◽

Transmembrane Segments ◽

Metal Binding Sites ◽

P Type

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Cross Talk between Sphingolipids and Glycerophospholipids in the Establishment of Plasma Membrane Asymmetry

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0458 ◽

2004 ◽

Vol 15 (11) ◽

pp. 4949-4959 ◽

Cited By ~ 69

Author(s):

Akio Kihara ◽

Yasuyuki Igarashi

Keyword(s):

Plasma Membrane ◽

Cross Talk ◽

Abc Transporters ◽

Transcriptional Factor ◽

Drug Efflux ◽

Lipid Asymmetry ◽

Membrane Asymmetry ◽

P Type ◽

Yeast Mutants ◽

Long Chain

Glycerophospholipids and sphingolipids are distributed asymmetrically between the two leaflets of the lipid bilayer. Recent studies revealed that certain P-type ATPases and ATP-binding cassette (ABC) transporters are involved in the inward movement (flip) and outward movement (flop) of glycerophospholipids, respectively. In this study of phytosphingosine (PHS)-resistant yeast mutants, we isolated mutants for PDR5, an ABC transporter involved in drug efflux as well as in the flop of phosphatidylethanolamine. The pdr5 mutants exhibited an increase in the efflux of sphingoid long-chain bases (LCBs). Genetic analysis revealed that the PHS-resistant phenotypes exhibited by the pdr5 mutants were dependent on Rsb1p, a putative LCB-specific transporter/translocase. We found that the expression of Rsb1p was increased in the pdr5 mutants. We also demonstrated that expression of RSB1 is under the control of the transcriptional factor Pdr1p. Expression of Rsb1p also was enhanced in mutants for the genes involved in the flip of glycerophospholipids, including ROS3, DNF1, and DNF2. These results suggest that altered glycerophospholipid asymmetry induces the expression of Rsb1p. Conversely, overexpression of Rsb1p resulted in increased flip and decreased flop of fluorescence-labeled glycerophospholipids. Thus, there seems to be cross talk between sphingolipids and glycerophospholipids in maintaining the functional lipid asymmetry of the plasma membrane.

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Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

10.1101/442459 ◽

2018 ◽

Cited By ~ 2

Author(s):

David M. Kern ◽

SeCheol Oh ◽

Richard K. Hite ◽

Stephen G. Brohawn

Keyword(s):

Lipid Bilayers ◽

Critical Role ◽

Anion Channel ◽

Lipid Binding ◽

Channel Structure ◽

Anion Channels ◽

Cryo Electron Microscopy ◽

Lipid Nanodiscs ◽

Insight Into

AbstractHypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

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Effect of nucleotides and environmental factors on the intermediate states of ATP hydrolysis cycle in skeletal muscle fibres

Hot Topics in Thermal Analysis and Calorimetry - The Nature of Biological Systems as Revealed by Thermal Methods ◽

10.1007/1-4020-2219-0_7 ◽

2006 ◽

pp. 159-186 ◽

Cited By ~ 1

Author(s):

D. ŐLõrinczy

Keyword(s):

Skeletal Muscle ◽

Environmental Factors ◽

Atp Hydrolysis ◽

Muscle Fibres ◽

Intermediate States ◽

Skeletal Muscle Fibres

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The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase

Science ◽

10.1126/science.abc5809 ◽

2020 ◽

Vol 369 (6511) ◽

pp. eabc5809 ◽

Cited By ~ 9

Author(s):

Michael J. McKenna ◽

Sue Im Sim ◽

Alban Ordureau ◽

Lianjie Wei ◽

J. Wade Harper ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Targeting ◽

Transmembrane Helix ◽

Protein Composition ◽

Binding Pocket ◽

Transmembrane Segment ◽

Substrate Binding Pocket ◽

Cryo Electron Microscopy ◽

Endogenous Substrate ◽

P Type

Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A–adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail–anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo–electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.

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